Plasmonic patch as a universal fluorescence enhancer

ABSTRACT

Fluorescence-based techniques are the cornerstone of modern biomedical optics with applications ranging from bioimaging at various scales (organelle to organism) to detection and quantification of a wide variety of biological species of interest. However, feeble fluorescence signal remains a persistent challenge in meeting the ever-increasing demand to image, detect and quantify biological species of low abundance. Disclosed herein are simple and universal methods based on a flexible and conformal elastomeric film adsorbed with plasmonic nanostructures, referred to as “plasmonic skin” or “plasmonic patch”, that provide large and uniform enhancement of fluorescence on a variety of surfaces, through an “add-on-top” process. The novel fluorescence enhancement approach presented here represents a disease-, biomarker-, and application-agnostic ubiquitously-applicable fundamental and enabling technology to improve the sensitivity of existing analytical methodologies in an easy-to-handle and cost-effective manner, without changing and/or minimally altering the original procedures of the existing techniques.

This application claims priority to U.S. Provisional Application62/590,877 filed on Nov. 27, 2017, which is incorporated by reference inits entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under grant CBET1254399awarded by the National Science Foundation, grants R21DK100759, RO1CA141521 and RO1 DE027098 awarded by the National Institutes of Health,and grant 3706 awarded by the Barnes-Jewish Hospital ResearchFoundation. The government has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

The field of this disclosure relates generally to fluorescenceenhancement. Specifically, it relates to the use of a novel plasmonicpatch comprising plasmonic nanoparticles attached to a flexiblesubstrate to enhance the fluorescence of a sample. Also disclosed hereinare methods that use this novel plasmonic patch to enhance thefluorescence signal in various biological assays.

Previous plasmon-enhanced fluorescence assays rely on engineering thebioassay surface to be plasmonically active through either deposition ofmetal islands or adsorption of plasmonic nanostructures. These methodsnaturally require the utilization of special surfaces and possiblysignificant alterations of the read-out devices and the bioassayprotocol. As such, they are not readily applied to a large variety ofsystems or bioassays.

Fluorescent probes and fluorometric approaches have been employed inbiomedical research, not only as imaging tools to visualize the locationand dynamics of cells, various sub-cellular species and molecularinteractions in cells and tissues, but also as labels influoroimmunoassays for detection and quantification of molecularbiomarkers. Fluorescence-based techniques have radically transformedbiology and life sciences by unravelling the genomic, transcriptomic,and proteomic signatures of disease development, progression, andresponse to therapy. However, “feeble signal” has been a persistent andrecurring problem in a battery of detection and imaging techniques thatrely on fluorescence. Overcoming this fundamental challenge without theuse of specialized reagents, equipment, or significant modifications towell-established procedures has been the subject of extensive researchin the field of biomedical optics. For example, there is an urgent needfor ultra-sensitive fluoroimmunoassays that are broadly adopted by mostbiological and clinical laboratories for the detection of targetbiological molecules of low abundance.

Improving the signal-to-noise ratio of the assays without radicallydeviating from existing assay protocols will also relax the stringentrequirements of high sensitivity and bulky photodetectors, drive downthe cost of implementation, eliminate cross-laboratory, cross-platforminconsistency, and propel these technologies to point-of-care, in-fieldand resource-limited settings. Various techniques, includingmultiple-fluorophore labels, rolling cycle amplification, and photoniccrystal enhancement have been introduced to improve the signal-to-noiseratio of fluorescence-based imaging and sensing techniques. Despite theimproved sensitivity, these technologies are not widely adopted inresearch and clinical settings. Most of these technologies requiresignificant modifications to the existing practices such as additionalsteps that significantly prolong the overall operation time, specializedand expensive read-out systems, non-traditional data processing andanalysis, or temperature-sensitive reagents which requiretightly-controlled transport and storage conditions.

Plasmon- or metal-enhanced fluorescence has been recognized as a simpleand highly effective approach for enhancing the bioanalytical parametersof fluorescence-based bioassays. Enhancement in the emission offluorophores in close vicinity to plasmonic nanostructures is attributedto the enhanced electromagnetic field (local excitation field) at thesurface of the plasmonic nanostructures and decrease in the fluorescencelifetime due to the coupling between excited fluorophores and surfaceplasmons of the nanostructures. So far, various plasmonic substratessuch as metal nano-islands have been shown to result in moderatefluorescence enhancement. Although these plasmonically active surfacesare attractive, they require the use of pre-fabricated specializedbioassay surfaces, typically a glass slide deposited with metalnanostructures, instead of standard or, sometimes, irreplaceablebioanalytical and bioimaging platforms. The requirement of specialbioassay surfaces limits cross-platform and cross-laboratory consistencyand seamless integration with widely employed bioanalytical procedures,which largely limits their extensive application in biomedical researchand clinical settings. Non-traditional bioconjugation procedures andpoor stability of biomolecules (e.g., antibodies) immobilized on metalsurfaces impose further challenges in their widespread application.Additionally, these plasmon-enhanced fluorescence assays can achievelower limits of detection than unenhanced assays, but the upper limitsof detection are also lower. This means that the dynamic range of thepreviously described plasmon-enhanced fluorescence techniques is, atbest, only marginally improved. Thus, quantification of high-abundanceanalytes is being sacrificed for quantification of low-abundanceanalytes.

Multiplexed microarrays based on fluorescence are employed in expressionprofiling, drug-target binding assays, and high throughput proteomics.Compared to a single platform such as an enzyme-linked immunosorbentassay (ELISA), this technique allows researchers and clinicians toexamine a large number of biomarkers in parallel to achieve patientstratification and monitoring of multifactorial diseases with limitedsample volume, thereby minimizing the assay cost and time to performmultiple individual biomarker assays. Moreover, high throughputprofiling of biomarkers enables personalized medicine with holistic,molecular fingerprinting of diseases, accommodating greater diagnosticresolution between closely related disease phenotypes. The sensitivityand specificity for diagnosis of kidney disease have been proven to besignificantly greater by combining the urinary levels of multiplebiomarkers than an individual one. However, despite the availability ofvarious commercialized products, this multiplexed methodology suffersfrom inferior sensitivity and relatively high limit of detection (LOD)compared to ELISA, which hinders its widespread application.

Thus, there is a need for addressing each of the disadvantages discussedherein, including low sensitivity, high dynamic range, cross-platformcompatibility, and ease of use.

BRIEF DESCRIPTION OF THE DISCLOSURE

In a first aspect, disclosed herein is a plasmonic patch for enhancing afluorescent signal from a fluorescent species. The patch generallycomprises: a flexible substrate comprising a first material, a plasmonicnanostructure, and a spacer comprising a second material. Thenanostructures are disposed on a first surface of the flexiblesubstrate, the spacer is disposed on the first surface of the flexiblesubstrate and covers the nanostructures; the fluorescent species has anexcitation wavelength (λ_(ex)), the plasmonic nanostructure has alocalized surface plasmon resonance (LSPR) wavelength (λ_(LSPR)), andthe difference between the LSPR wavelength and the excitation wavelengthis |Δλ|.

In a second aspect, disclosed herein is a method for enhancing afluorescent signal of a fluorescent species. The method generallycomprises: placing a plasmonic patch in proximity to the fluorescentspecies, exciting the fluorescent species using electromagneticradiation of a predetermined wavelength thereby generating thefluorescent signal, and detecting said enhanced fluorescent signal. Theplasmonic patch generally comprises: a flexible substrate comprising afirst material, a plasmonic nanostructure, and a spacer comprising asecond material. The nanostructures are disposed on a first surface ofthe flexible substrate, the spacer is disposed on the first surface ofthe flexible substrate and covers the nanostructures; the fluorescentspecies has an excitation wavelength (λ_(ex)), the plasmonicnanostructure has a localized surface plasmon resonance (LSPR)wavelength (λ_(LSPR)), and the difference between the LSPR wavelengthand the excitation wavelength is |Δ∥|.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an exemplary embodiment of a schematic illustration of thefabrication of plasmonic patch and its application in fluoroimmunoassaythrough a simple “add-on-top” process, which results in largeenhancement in the fluorescence signal. Right inset (top): photographshowing the transfer of a plasmonic patch to a planar surface. Rightinset (middle): SEM image demonstrating the flexibility as well asconformability of the plasmonic patch to the surface. Right inset(bottom): SEM image of the cross-section of plasmonic patch showing anaverage thickness of 30 μm.

FIG. 2 is an exemplary embodiment of the normalized extinction spectraof the aqueous solutions of three representative plasmonicnanostructures in accordance with the present disclosure (from left toright: Au@Ag-490, AuNR-670, AuNR-760). The extinction bands ofAu@Ag-490, AuNR-670, and AuNR-760 exhibit significant overlap with theabsorption bands (excitation source) of FITC, LT680, and 800CW,respectively indicated with three different shades.

FIG. 3 depicts exemplary embodiments of SEM images of a plasmonic patchsurface revealing the uniform distribution of plasmonic nanostructureson a polymer film (from left to right: Au@Ag-490, AuNR-670, AuNR-760) inaccordance with the present disclosure. Insets show the representativeTEM images of the corresponding plasmonic nanostructures.

FIG. 4 is an exemplary embodiment of an extinction spectra of plasmonicpatches adsorbed with distinct plasmonic nanostructures (measured inair) in accordance with the present disclosure.

FIG. 5 is an exemplary embodiment of a fluorescence map of threefluorophores adsorbed on silicon substrate in the presence and absenceof plasmonic patch in accordance with the present disclosure.

FIG. 6 depicts the transfer process of and ease of handling of plasmonicpatch (AuNR-670) using a sharp forceps in accordance with the presentdisclosure. Scale bar=5 mm.

FIGS. 7A, 7B and 7C are exemplary embodiments of fluorescence spectra of(7A) FITC, (7B) LT680, and (7C) 800CW adsorbed on silicon substrate inthe presence and absence of plasmonic patch in accordance with thepresent disclosure.

FIG. 8 is an exemplary embodiment of a fluorescence enhancement ofplasmonic patch of 800CW uniformly adsorbed on silicon, glass,nitrocellulose, and polystyrene surfaces in accordance with the presentdisclosure. Circular plasmonic patch with AuNR-760 (with a diameter of 4mm) is applied on each surface coated with 800CW. Fluorescent intensityprofile across the plasmonic patch reveals strong enhancement within theplasmonic patch region compared to the area outside the film (75 timesfor silicon, 24 times for glass, 9 times for nitrocellulose, and 80times for polystyrene).

FIG. 9 is an exemplary embodiment of a schematic illustration showing aplasmonic patch with a polymer layer, acting as the spacer betweenfluorophores and plasmonic nanostructures in accordance with the presentdisclosure. Spacer thickness is adjusted to achieve different amounts ofenhancement efficiency.

FIG. 10 is an exemplary embodiment of a schematic illustration showingthe copolymerization of TMPS and APTMS, which is employed as a spacerlayer on plasmonic patch, in accordance with the present disclosure.

FIGS. 11A and 11B are exemplary embodiments of: a (11A) LSPR wavelengthof pristine AuNR and AuNR coated with siloxane copolymer; TMPS/APTMS(4/4) employed in the polymerization process from left to right: 0:0,0:8, 0.25:8, 0.5:8, 1:8, 4:8, and 8:8; and, a (11B) Red-shift in LSPRwavelength (compared to pristine AuNR) as a function of amount of TMPSemployed during polymerization (with APTMS amount set to 8 μL) inaccordance with the present disclosure.

FIG. 12 is an exemplary embodiment of a fluorescence map of 800CW in thepresence of plasmonic patch with increasing spacer layer thickness (TMPSand APTMS volume ratio is 0:0, 0:8, 0.25:8, 0.5:8, 1:8, 4:8, 8:8 fromleft to right) in accordance with the present disclosure.

FIG. 13 is an exemplary embodiment of a fluorescence enhancement factoras a function of spacer thickness (TMPS to APTMS ratio in polymerizationprocess) in accordance with the present disclosure.

FIG. 14 is an exemplary embodiment of AFM images of pristine Au nanorods(left) and Au nanorods with polymer spacer (right) (TMPS and APTMSvolume ratio of 4:8) in accordance with the present disclosure.

FIG. 15 is an exemplary embodiment of a schematic showing the concept ofplasmonic patch enhanced fluoroimmunoassay implemented in a glass bottom96-well plate, demonstrating the wide applicability of the plasmonicpatch in accordance with the present disclosure.

FIGS. 16A and 16B are exemplary embodiments of fluorescence intensitymaps of fluoroimmunoassay corresponding to different concentrations of(16A) Kidney Injury Molecule-1 (KIM-1) and (16B) Neutrophil GelatinaseAssociated Lipocalin (NGAL), two early stage biomarkers for acute kidneyinjury (AKI) and chronic kidney disease (CKD) (Left and middle imagesshow the unenhanced assays which correspond to different color scalesshown in the figures. Right image shows the plasmonic patch enhancedassay revealing a large enhancement in the fluorescence signal as wellas a broadened dynamic range compared to unenhanced assay (scale barrepresents 5 mm)) in accordance with the present disclosure.

FIG. 17 is an exemplary embodiment of a fluorescence intensity of aplasmonic patch enhanced fluoroimmunoassay after being stored with theplasmonic patch for 2, 4, 6, and 8 weeks in accordance with the presentdisclosure. Unlike colorimetric ELISA that shows rapid degradation inthe optical intensity, plasmonic patch-enhanced fluoroimmunoassayexhibits a stable signal with no compromise in the LOD for up to 8weeks.

FIGS. 18A and 18B are exemplary embodiments of plots showing thefluorescence intensities corresponding to different concentrations of(18A) MM-1 and (18B) NGAL in accordance with the present disclosure. Thelimits of detection identified in the plots show ˜300-fold and ˜100-foldimprovement for MM-1 and NGAL, respectively, compared to the unenhancedassay.

FIGS. 19A and 19B are exemplary embodiments of: (19A) fluorescenceintensity maps of NGAL fluoroimmunoassay implemented on a common 96-wellplate with a polystyrene bottom (left and middle images show theunenhanced assays which correspond to different color scales shown inthe images, right image shows the plasmonic patch enhanced assayrevealing a large enhancement in the fluorescence signal (scale barrepresents 5 mm)); and, (19B) a plot showing the calibration curve forplasmonic patch enhanced fluoroimmunoassay implemented on 96-well platewith a polystyrene bottom in accordance with the present disclosure.

FIG. 20 is an exemplary embodiment of a MM-1 concentration-responsecurve obtained by ELISA in accordance with the present disclosure. TheLOD is determined to be 15.6 pg/ml.

FIGS. 21A and 21B are exemplary embodiments of fluorescence intensitymaps of: (21A) KIM1 (10-fold dilution); and, (21B) NGAL (40-folddilution) immunoassay for urine samples from eight patients and threeself-described healthy volunteers in accordance with the presentdisclosure. Top: Unenhanced fluorescence map. Bottom: Plasmonic patchenhanced fluorescence map. Scale bar=5 mm.

FIG. 22 depicts exemplary embodiments of graphs of the MM-1 (22A) andNGAL (22B_ concentrations in the urine samples (diluted 10-fold forKIM-1 and 40-fold for NGAL) as determined by unenhanced fluorescenceassay, plasmon-enhanced fluorescence assay, and ELISA in accordance withthe present disclosure.

FIG. 23 depicts exemplary embodiments of graphs showing the correlationbetween the concentration of (23A) KIM-1 and (23B) NGAL determined usingELISA and plasmonic patch enhanced fluorescence assay in accordance withthe present disclosure.

FIG. 24 illustrates an exemplary embodiment of an application ofplasmonic patch to enhance the bioanalytical parameters of a proteinmicroarray in accordance with the present disclosure. Left: Photographdepicting the plasmonic patch employed for enhancing the fluorescence ofprotein microarray (scale bar represents 5 mm), SEM image demonstratingthe uniform absorption of AuNR-760 on the PDMS surface (scale barrepresents 500 nm), and photograph of commercial protein microarraysubstrate with 16 sub-wells for simultaneous analysis of multiplesamples (scale bar represents 1 cm). Right: Schematic showing theconcept of plasmonic patch enhanced microarray, which enables a highlysensitive profiling of eight AKI and CKD biomarkers, simultaneously.

FIG. 25 depicts exemplary embodiments of three schematics illustratingthe layout of antibodies printed on the bottom of each sub-well (left),the unenhanced fluorescence intensity map representing the AKI and CKDprotein biomarker profile of patient #81 (middle), and a fluorescencemap generated after the application of plasmonic patch (scale barrepresents 400 μm) (right) in accordance with the present disclosure.

FIG. 26 is an exemplary embodiment of an unenhanced (top) and plasmonicpatch enhanced (bottom) fluorescence intensity maps representing thekidney disease biomarker profile of patients and healthy volunteers(scale bar=400 μm) in accordance with the present disclosure.

FIG. 27 depicts exemplary embodiments of quantitative measurements offluorescence intensity corresponding to the concentrations of variousbiomarkers in the urine samples of four patients and two self-describedhealthy volunteers in accordance with the present disclosure. The [+]indicates biomarker detected only after the application of the plasmonicpatch. POS spots in the microarray represent three distinct PositiveControl Signal intensities (POS1>POS2>POS3) of a biomarker unrelated tokidney disease.

FIG. 28 depicts an exemplary embodiment of a fluorescence intensity heatmap corresponding to the concentrations of kidney diseases biomarkers inthe urine samples of four patients and two healthy volunteers before(top) and after (bottom) applying the plasmonic patch in accordance withthe present disclosure.

FIGS. 29A to 29D depict (29A) fluorescence maps obtained from sandwichassay wells (cTnI concentration of 4000 pg/mL) without and with patchshowing a progressive increase in the fluorescence intensity withincreasing AuNR-758 density (29B) Plot showing the fluorescenceenhancement factor of plasmonic patches described in (29B). (29C)Fluorescence map (top) and intensity (bottom) of cTnI fluoroimmunoassayon 96-well plate without plasmonic patch. (29D) Fluorescence map (top)and intensity (bottom) of cTnI fluoroimmunoassay on 96-well plate withplasmonic patch, showing a limit-of-detection of 30 μg/mL.

FIGS. 30A to 30G depict (102A) TEM image of AuNR-758. (30B) Extinctionspectra of plasmonic patches comprised of different densities ofAuNR-758 after being transferred to 96 well plate. (30C) SEM images ofthe plasmonic patch surface revealing the uniform distribution ofAuNR-758 with increasing density. (30D and 30F) Fluorescence mapswithout and with patch showing a progressive increase in thefluorescence intensity with increasing AuNR density (30E and 30G) Plotshowing the fluorescence enhancement factor of the plasmonic patches asfunction of the density of AuNR.

FIGS. 31A to 31D depict (31A) SEM images of the top surfaces ofplasmonic patches modified with AuNR of various size (length/diameter).The densities of AuNRs were controlled to be similar between differentplasmonic patches. (31B) Fluorescence map showing the enhancement ofemission of 800CW using AuNR of different size. (31C) Plot showing thefluorescence enhancement factors using these AuNRs. (31D) LSPRwavelength of plasmonic patch after being transferred to 96-well plate(recorded by plate reader), showing a slight red shift compared to theLSPR wavelength measured in aqueous solutions.

FIGS. 32A to 32C depict (32A) TEM images of gold nanorods (AuNRs)employed as the nanoantennae on plasmonic patch, showing differentlength and diameter (nm). (32B) Normalized extinction spectra of theaqueous suspensions of different size AuNRs showing similar LSPRwavelength. (32C) The aspect ratio (length:diameter) of each AuNR.

FIGS. 33A and 33B depict (33A) the fluorescence signal of a conventionalIL-6 (from 6 fg/mL to 6 ng/mL) fluoroimmunoassay (top) and an exemplaryembodiment of the plasmonic patch enhanced IL-6 fluoroimmunoassay(middle: AuNR 130 nm; Bottom: AuNR 57 nm). (33B) Plot showing thefluorescence intensity at different analyte concentration. Thelimit-of-detection of plasmonic patch enhanced fluoroimmunoassay usingAuNR-130 nm is calculated to be 0.01 μg/mL, which is 200-fold lowercompared to AuNR-57 nm (2 μg/mL).

FIG. 34 illustrates one exemplary embodiment of the plasmonic patch thatcomprises a rigid backer and a reflective layer in addition to theflexible substrate, spacer layer and plasmonic nanoparticles.

DETAILED DESCRIPTION OF THE DISCLOSURE

In the following specification and the claims, reference will be made toa number of terms, which shall be defined to have the followingmeanings. The singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. The terms“comprising,” “including,” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements. “Optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where the event occurs and instanceswhere it does not.

Approximating language, as used herein throughout the specification andclaims, may be applied to modify any quantitative representation thatcould permissibly vary without resulting in a change in the basicfunction to which it is related. Accordingly, a value modified by a termor terms, such as “about,” “approximately,” and “substantially,” are notto be limited to the precise value specified. In at least someinstances, the approximating language may correspond to the precision ofan instrument for measuring the value. Here and throughout thespecification and claims, range limitations may be combined and/orinterchanged; such ranges are identified and include all the sub-rangescontained therein unless context or language indicates otherwise.

The plasmonic patch disclosed herein overcomes the above-mentionedchallenges and provides a path forward for broad application ofsensitive, high dynamic range multiplexed microarrays. In one example,the detection of biomarkers related to kidney function, the resultsillustrate that the plasmonic patch significantly enhances the abilityto elucidate low-level kidney function parameters (biomarkers) toprovide holistic kidney disease information. Notably, the betterperformance of the multiplexed microarray emanates from the extremelysimple “plasmonic patch transfer” process, which does not alter theestablished process flow of immuno-microarrays. Additionally, thistechnique represents an inexpensive and easily implemented approach forthe enhancement of fluorescence. This easy-deployable technique isseamlessly applied to a broad range of multiplexed platforms indiagnostics, proteomics, and genetics to address the unmet need forhigher signal intensity. The Examples provided herein are onlyillustrative, not limiting.

Most previous plasmon-enhanced fluorescence assays rely on engineeringthe bioassay surface to be plasmonically active through eitherdeposition of metal islands or adsorption of plasmonic nanostructures.These methods naturally require the utilization of special surfaces andpossibly significant alterations of the read-out devices and thebioassay protocol. Demonstrated herein is a novel method in which theenhancement is achieved by simple transfer of plasmonic patch onto asurface with fluorescent species. This novel approach obviates the needfor special bioassay surfaces or tedious bioconjugation procedures andoffers excellent tunability of the plasmonic properties (over the entirevisible and IR wavelength range) and distance between the metal surfaceand fluorophores. Notably, the magnitude of fluorescence enhancementusing plasmonic surfaces described in the past is highly dependent onthe size of the capture antibody, antigen, and detection antibody thatexist between the plasmonic nanostructures on the surface and thefluorophores. The enhancement is therefore dictated by the preset“biological spacer”, leaving little control over one of the key designparameter for maximum enhancement-spacer layer thickness. On thecontrary, as an “add-on-top” layer, the plasmonic patch disclosed hereinenables complete control over the distance between plasmonicnanostructures and fluorescent species. The facile control of the spacerthickness provides a tunable fluorescence enhancement despite thevariations in the immunofluorescent assays, which is especiallyimportant in multiplexed platforms.

In some aspects, the fluoroimmunoassay is read before and after additionof the plasmonic patch. The combined pre- and post-patch additionmeasurements provide an extraordinary dynamic range in most bioassays.Analytes of low abundance are not detectable without addition of thepatch, whereas the signal from high abundance analytes saturates thedetector in the presence of the patch. The upper limit of detection ofthe fluoroimmunoassay remains the same as the unenhancedfluoroimmunoassay because the fluoroimmunoassay is read before addingthe patch. The lower limit of detection is decreased because the assayis read after adding the patch. This increases the overall dynamic rangeof the fluoroimmunoassay by from 2 to 4 orders of magnitude. Thus,reading a fluoroimmunoassay before and after adding the plasmonic patchresults in an overall dynamic range of from about 5 to 7 orders ofmagnitude.

In some embodiments, disclosed herein is a plasmonic patch that isplaced on top of a sample to be analyzed to thereby enhance thefluorescence emanating from the sample. The plasmonic patch comprises: aflexible substrate comprising a first material, a plasmonicnanostructure, and a spacer comprising a second material. Thenanostructures are disposed on a first surface of the flexiblesubstrate; the spacer is disposed on the first surface of the flexiblesubstrate and covers the nanostructures; the fluorescent species has anexcitation wavelength (λ_(ex)), the plasmonic nanostructure has alocalized surface plasmon resonance (LSPR) wavelength (λ_(LSPR)), andthe difference between the LSPR wavelength and the excitation wavelengthis |Δλ|. In some embodiments, the plasmonic patch is in the form of athin film or skin.

As used herein, the term “conformable” refers to a property of thesubstrate such that when the substrate is placed on top of anothersurface (e.g., a microtiter plate or glass slide), the shape of thesubstrate alters or substantially conforms to and takes the shape of thesurface on which it is placed. A substantial amount of contact betweenthe substrate and the surface is presumed. In some aspects, theplasmonic patch has a hardness as measured on the Shore 00 scale of lessthan 90, less than 80, less than 70, less than 60, less than 50, lessthan 40, less than 30, less than 25, less than 20 or even less than 15.In some aspects, the Shore A hardness is less than 0. In some aspectsdisclosed herein, the plasmonic patch is conformable and will take theshape of a surface on which it is placed.

As used herein, the term “cover” or “covers” in reference to therelationship between the spacer and the plasmonic nanoparticles refersto the manner in which the material that comprises the spacer covers theplasmonic nanoparticles. In some aspects, the nanoparticles arecompletely encompassed by the spacer material such that they are notexposed to the atmosphere or outer environment. In some aspects, thenanoparticles are only partially encompassed by the spacer materials anda portion of the surface of the nanoparticle is exposed to theatmosphere and outer environment. In some aspects, only thenanoparticles are covered with a spacer while the flexible substrate isnot covered.

The plasmonic patch disclosed herein is useful for enhancing thebioanalytical parameters (sensitivity, LLOD, and dynamic range) offluoroimmunoassays implemented in a microplate format and an antibodymicroarray. In some embodiments, the microplate is in the form of astandard 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, or1536-well plate. In other embodiments, the format of the immunoassay ison a glass slide or other formats as are known in the art. In someembodiments, the plasmonic patch results in more than about a 10-fold,about a 25-fold, about a 50-fold, about a 75-fold, about a 100-fold,about a 150-fold, about a 200-fold, about a 300-fold, about a 400-fold,about a 500-fold, about a 750-fold, about a 1000-fold, about a2500-fold, about a 5000-fold, about a 7500-fold, or about a 10,000-foldfluorescence intensity enhancement. An increase in the fluorescenceintensity enhancement leads to an increase in the assay sensitivity anda lower LOD. In some embodiments, the lower LOD decreases by more thanabout 10-fold, about 25-fold, about 50-fold, about 75-fold, about100-fold, about 150-fold, about 200-fold, about 300-fold, about400-fold, about 500-fold, about 750-fold, about 1000-fold, about2500-fold, about 5000-fold, about 7500-fold, or about 10,000-fold.Additionally, this increases the overall dynamic range of detection. Insome embodiments, the increase in the overall dynamic range is greaterthan one order of magnitude, two orders of magnitude, three orders ofmagnitude or even four orders of magnitude.

The improvement in the bioanalytical parameters was found to beconsistent across different assay formats, target biomarkers, andfluorophores. Significantly, this method is implemented with existingbioassays without any modification of the standard operating procedures,additional operational training, or modification of the read-outdevices. Additionally, in some aspects, the method described herein isused on whole cells, tissues, and/or glass slides. As a part of rigorousvalidation of the technology, urine samples from patients and healthyvolunteers have been analyzed. As opposed to unenhancedfluoroimmunoassay and ELISA, the plasmon-enhanced fluoroimmunoassayenabled the detection and quantification of low concentrationbiomarkers, and from all patients and healthy volunteers. The addedsensitivity of the plasmon-enhanced assay enables the facilequantification of biomarkers of low abundance and provides physiologicaland pathological information, often missed by the conventionalimmunoassays.

The first and the second material used to construct the plasmonic patchare the same or different. The material has specific characteristics inorder to give optimal results. The first material has a high mechanicalflexibility. In some aspects, the second material is less flexible thanthe first material. In some aspects, the Shore 00 hardness of the secondmaterial is greater than the Shore 00 hardness of the first material. Insome aspects, the first and/or second material have high opticaltransparency. Any material exhibiting these characteristics andcompatible with the nanostructures is suitable for use herein. Othercharacteristics of the material are selected based on the specificapplication and knowledge of the person of skill in the art. In someaspects, the first material and the second material, independently fromone another, comprise a polymer. In some aspects, the first material isan elastomeric polymer. In some aspects, the first and the secondmaterial are functionalized with siloxane. In some aspects, the firstand second material are, independently from one another, selected fromthe group consisting of polydimethylsiloxane (PDMS), ECOFLEX®,SILBIONE®, polyethylene terephthalate (PET), polyurethane (PU),polyethylene naphthalate (PEN), polyimide (PI), polybutadiene,polyisoprene, (3-aminopropyl)trialkoxysilane,(3-aminopropyl)triaryloxysilane, (3-aminopropyl)triethoxysilane (APTES),(3-aminopropyl)trimethoxysilane (APTMS), trimethoxy(propyl)silane(TMPS), (3-mercaptopropyl)trimethyoxysilane (MPTMS), polyamine,poly(methyl)methacrylate (PMMA), polydopamine, polyvinylpyrrolidinone(PVP), polyvinyl alcohol (PVA), polyolefin, polyamide, polyimide,proteins, silk, cellulose, polyelectrolytes, peptoids and combinationsthereof. In some aspects, the first material is PDMS. In some aspects,the first material is ECOFLEX®. Additionally, the spacer may comprisematerial selected from the group consisting of silicon oxide, aluminumoxide, zinc oxide, titanium oxide, graphene, graphene oxide, MoS₂,MXenes, and combinations thereof.

The thickness and refractive index of the spacer is affected by, atleast in part, the composition of the second material. In some aspects,the second material is APTES, APTMS, TMPS, MPTMS, or a combinationthereof. In some aspects, the second material comprises from about 0 toabout 100 wt % of APTES. In some aspects, the second material comprisesabout 5 wt % APTES, about 10 wt % APTES, about 15 wt % APTES, about 20wt % APTES, about 25 wt % APTES, about 30 wt % APTES, about 35 wt %APTES, about 40 wt % APTES, about 45 wt % APTES, about 50 wt % APTES,about 55 wt % APTES, about 60 wt % APTES, about 65 wt % APTES, about 70wt % APTES, about 75 wt % APTES, about 80 wt % APTES, about 85 wt %APTES, about 90 wt % APTES, or about 95 wt % APTES. As used in thiscontext, about means±2.5 wt %.

In some aspects, the second material comprises from about 0 to about 100wt % of TMPS. In some aspects, the second material comprises about 5 wt% TMPS, about 10 wt % TMPS, about 15 wt % TMPS, about 20 wt % TMPS,about 25 wt % TMPS, about 30 wt % TMPS, about 35 wt % TMPS, about 40 wt% TMPS, about 45 wt % TMPS, about 50 wt % TMPS, about 55 wt % TMPS,about 60 wt % TMPS, about 65 wt % TMPS, about 70 wt % TMPS, about 75 wt% TMPS, about 80 wt % TMPS, about 85 wt % TMPS, about 90 wt % TMPS, orabout 95 wt % TMPS. As used in this context, about means±2.5 wt %.

In some aspects, the second material comprises from about 0 to about 100wt % of MPTMS. In some aspects, the second material comprises about 5 wt% MPTMS, about 10 wt % MPTMS, about 15 wt % MPTMS, about 20 wt % MPTMS,about 25 wt % MPTMS, about 30 wt % MPTMS, about 35 wt % MPTMS, about 40wt % MPTMS, about 45 wt % MPTMS, about 50 wt % MPTMS, about 55 wt %MPTMS, about 60 wt % MPTMS, about 65 wt % MPTMS, about 70 wt % MPTMS,about 75 wt % MPTMS, about 80 wt % MPTMS, about 85 wt % MPTMS, about 90wt % MPTMS, or about 95 wt % MPTMS. As used in this context, aboutmeans±2.5 wt %.

The nanostructures used herein provide the plasmonic enhancement to thefluorescent signal and are selected based on numerous criteria. Onecriterion in selecting the nanostructure material to use in theplasmonic patch is the localized surface plasmon resonance wavelength(LSPR wavelength or λ_(LSPR)). As used herein, the term λ_(LSPR) isdefined to be any wavelength that excites plasmons in thenanostructures. Different nanostructures have a different λ_(LSPR) asillustrated in FIG. 2. Because λ_(LSPR) can be selected or tuned in thenanostructures in part by the method in which they are prepared, theλ_(LSPR) is selected for a given plasmonic patch based on the excitationwavelength (λ_(ex)) of the fluorescent species or the emissionwavelength of the fluorescent species (λ_(em)). In some aspects, theλ_(ex) or λ_(em) is either a local maximum or the global maximum for thefluorescent species.

Additionally, the λ_(LSPR) of a nanostructure shifts after incorporationinto the plasmonic patch by being encapsulated in the spacer layer andby being placed into proximity of the bioassay surface comprising afluorescent species. Without being bound by a specific theory, it isthought that the change in the index of refraction of the mediumsurrounding the plasmonic nanostructure causes the shift in theλ_(LSPR). As such, the properties of the second material which comprisesthe spacer layer that covers and encapsulates the nanostructures willaffect the λ_(LSPR). Unless clearly stated otherwise, the λ_(LSPR) valuereferenced herein for the nanostructure is after the nanostructure hasbeen incorporated into the plasmonic patch, covered by the spacer layer,and brought into proximity of the bioassay surface.

The difference between the LSPR wavelength and the excitation wavelengthis |Δλ|:

|Δλ|=|Δ_(LSPR)−λ_(ex)|

and, in some aspects, the smaller |Δλ| (the absolute value of thedifference between λ_(LSPR) and λ_(ex)), the greater the enhancement ofthe fluorescent signal. In some aspects, |Δλ| is less than 50 nm, lessthan 45 nm, less than 40 nm, less than 35 nm, less than 30 nm, less than25 nm, less than 20 nm, less than 15 nm, less than 10 nm, less than 5nm, less than 2 nm, or less than 1 nm. In some aspects, the λ_(LSPR) andthe λ_(ex) are the same, meaning |Δλ| is zero. In this instance, zero isdetermined based on the accuracy and limitations of the instrumentation.

The specific λ_(LSPR) is based upon the λ_(ex). This allows for theplasmonic patch to be selectively tuned to match the weak fluorescencesignal to which it is applied. In some embodiments, the λ_(LSPR) isbetween about 200 and about 2000 nm, between about 250 and about 1500nm, between about 300 and about 1000 nm, between about 350 and about 800nm, between about 400 and about 750 nm. In yet another embodiment, theλ_(LSPR) is approximately (meaning ±25 nm) 300 nm, 350 nm, 400 nm, 450nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900nm, 950 nm, 1000 nm, 1100, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm,1700 nm, 1800 nm, 1900 nm, or 2000 nm. Suitable examples ofnanostructures include, but are not limited to, nanotubes, nanorods,nanocubes, nanocuboids, nanospheres, bimetallic nanostructures (e.g.Au@Ag core-shell nanocuboids), nanostructures with sharp tips (e.g.nanostars), hollow nanostructures such as nanocages and nanorattles,nanobipyramids, nanoplates, self-assembled nanostructures, bowtieantennae, nanoraspberries, nano islands and combinations thereof. Insome aspects, the plasmonic nanostructures are nanorods. In someaspects, the plasmonic nanostructures are nanocubes, nanocuboids or acombination thereof.

Nanorods can be fabricated in a large array of sizes and dimensions. Insome aspects, the nanorods have a length of from 25 to 1,000 nm (1 μm).In some aspects, the nanorods have a length of from 40 to 180 nm. Insome aspects, the nanorods have a length of from 60 to 160 nm. In someaspects, the nanorods have a length of from 80 to 140 nm. In someaspects, the nanorods have a length of from 100 to 130 nm. In someaspects, the nanorods have a length of about 40 nm, about 50 nm, about60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 250nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000nm, about 1200 nm, about 1400 nm, about 1600 nm, about 1800 nm, about2000 nm. In the same way that the length of the nanorods may vary, thediameter may also vary. In some aspects, the diameter of the nanorods isfrom about 4 nm to about 100 nm, from about 5 nm to about 80, from about20 nm to about 60 nm, or from about 25 nm to about 40 nm. In someaspects, the diameter of the nanorods is about 5 nm, about 10 nm, about15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm,about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm,or about 100 nm.

Nanocubes and nanocuboids can also be fabricated in a large array ofsizes, as described in Liu, et al., Chem. Mater., 27, 5261 (2015) whichis incorporated by reference for its teachings thereof. In some aspects,the average edge length is from about 25 nm to about 1000 nm (1 μm),from about 40 nm to about 1000 nm, from about 45 nm to about 750 nm,from about 50 nm to about 500, from about 55 nm to about 250 nm, or fromabout 60 nm to about 200 nm. In some aspects, the average length isabout 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm,about 80 nm, about 85 nm, about 90 nm, about 65 nm, about 100 nm, about110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about175 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm about 600nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.

The plasmonic nanostructures can be fabricated from any plasmonicmaterials, including, but not limited to, gold, silver, aluminum,copper, semiconductor materials, and combinations thereof. In someaspects, the material is gold. In some aspects, the material is silver.In some aspects, the material is both gold and silver. In still yetanother aspect, the material is a silver core encompassed by a goldshell. In still yet another aspect, the material is a gold coreencompassed by a silver shell. As used herein, a plasmonic material isone that exhibits oscillations of electrons upon excitation.

The density of the plasmonic nanostructures in the plasmonic patchaffects the amount of enhancement of the fluorescent signal from thefluorescent species. In some aspects, in order to provide a more uniformenhancement, the plasmonic nanostructures are distributed in an apparentmonolayer. As such, the density is of the nanostructures in the patch isa function of the area rather than the volume. In some aspects, thedensity of the nanostructures is from about 1/μm² to about 150/μm², fromabout 5/μm² to about 125/μm², about 10/μm² to about 100/μm², about20/μm² to about 75/μm², about 30/μm² to about 60/μm², or about 40/μm² toabout 60/μm². In some aspects, the density of the nanostructures isabout 10/μm², about 20/μm², about 30/μm², about 40/μm², about 50/μm²,about 60/μm², about 70/μm², about 80/μm², about 90/μm², about 100/μm²,about 110/μm², about 120/μm², about 130/μm², about 140/μm², about150/μm², about 160/μm², about 170/μm², about 180/μm², about 190/μm², orabout 200/μm².

The plasmonic patch disclosed herein is suitable for use with any assaythat uses fluorescence detection of the analytes. Examples of assaysthat are suitable for use herein include, but are not limited to,antibody/protein microarrays, bead/suspension antibody/proteinmicroarrays, arrays, bead suspension arrays, biosensing, biochip assays,capillary/sensor arrays, cell assays, DNA microarrays/polymerase chainreaction (PCR)-based arrays, energy transfer-based arrays, glycan/lectinarrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-basedarrays, microfluidic chips, reversed-phase protein arrays,protein/immunological assays, chemical compound arrays, and tissuearrays.

Additionally, the plasmonic patch disclosed herein is suitable forenhancing a fluorescent signal from a large variety of differentfluorescent sources or species. In addition to the fluorescent assaysdisclosed elsewhere herein, the plasmonic patch enhances the fluorescentsignal from fluorescent proteins, organic dyes, quantum dots andupconversion nanoparticles.

The thickness of the spacer is tunable thereby providing differentlevels of enhancement of a fluorescent signal in a fluorescent species.In some embodiments, the thickness of the spacer is from about 0.5 nm toabout 100 nm or from about 0.5 nm to about 20 nm. In yet otherembodiments, the thickness of the spacer is approximately 0.5 nm, 1 nm,2 nm, 3 nm, 5 nm, 8 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm,45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm or 100 nm. Approximately as used here means±10% of the giventhickness. The thickness of the spacer is controlled by increasing theconcentration and/or the amount of the monomers during preparation. Anypolymer that can be deposited uniformly across the plasmonicnanostructures, such as those described elsewhere herein, is used inaccordance with the present disclosure. In some aspects, polymer spacersare added in a layer-by-layer process, as in, for example, Feng, et al.(Sci. Reports, 5(7779), 1 (2015)) and Lakowicz, et al. (J. Flouresc.,14(4), 425 (2004)) which are incorporated by reference herein for theirteachings thereof. Additionally, inorganic spacers such as silica,alumina and/or zinc oxide are used. In some embodiments, they aredeposited on the plasmonic nanostructures using physical or chemicalvapor deposition or atomic layer deposition.

In some aspects, the refractive index of the spacer is varied in orderto shift the λ_(LSPR) of the nanostructures. In some aspects, therefractive index of the spacer is between about 0.8 and 2.0. In someaspects, the refractive index of the spacer is about 1.0, about 1.1,about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about1.8, about 1.9, or about 2.0. In this context, about means±0.05.

Elastic modulus is a quantity that measures the resistance of an objectto being deformed in an elastic (i.e., non-permanent) manner when stressis applied to the object. It is defined as the slope of thestress-strain curve in the elastic deformation region of the graph. Ahigher elastic modulus will be more rigid and more resistant todeformation. In some aspects, the flexible substrate that comprises thefirst material is an elastic polymer. In some aspects, the elasticmodulus of the flexible substrate is less than 50 MPa, less than 40 MPa,less than 30 MPa, less than 20 MPa, less than 15 MPa, less than 10 MPa,less than 7.5 MPa, less than 5 MPa, less than 4.5 MPa, less than 4 MPa,less than 3.5 MPa, less than 3 MPa, less than 2.5 MPa, less than 2 MPa,less than 1.5 MPa, or less than 1 MPa. In some aspects, the substrate isconformable, flexible or both.

In some exemplary embodiments, the plasmonic patch further comprises abacking layer. The backing layer is there to increase the rigidity ofthe patch and help facilitate handling of the patch. In some aspects,the backer layer has a higher Shore 00 hardness than that of theflexible substrate that comprises the first material. When present, thebacking layer is disposed on one side of the flexible substrate whilethe nanostructures and spacer is disposed on the other. In other words,the backing layer, the flexible substrate and the spacer form a threelayer system with the flexible substrate in the middle. When present,the backing layer comprises a material selected from the groupconsisting of glass, plastic, a polymer, aluminum, mylar, RE1SIAR™ (madeby 3M™), and combinations thereof.

In some exemplary embodiment, the plasmonic patch further comprises areflective layer. The reflective layer is present to further enhance thefluorescent signal from the fluorophore. The reflective layer isdisposed between the backing layer and the spacer. In some aspects, thereflective layer is disposed directly in contact with the backing layer.In still yet another aspect, the reflective layer is disposed directlybetween the backing layer and the flexible substrate. In some aspects,the reflective layer comprises a deposited metal such as, for example,aluminum, gold, silver and/or chromium. The reflective layer may bedeposited using, for example, sputter coating or vapor deposition. Insome aspects, the backer layer is the same as the reflective layer.

In preparing the plasmonic patch, the plasmonic nanostructures areattached to, or embedded in the first material in the flexiblesubstrate. They can be attached to the first material covalently, viahydrophobic interaction, via electrostatic interaction, via polarinteraction, via physisorption or a combination thereof. In someaspects, the attachment is covalent. In some aspects, the attachment isvia a hydrophobic interaction. In some aspects, the attachment is via anelectrostatic interaction. In some aspects, the attachment is viaphysisorption. In some aspects, the attachment is via polarinteractions. In some aspects, the attachment uses a combination of twoor more of attachment mechanisms.

In some aspects, the plasmonic nanoparticles are attached to theflexible substrate via electrostatic interaction. As an non-limitingexample, the first material is activated using an oxygen plasmatreatment and then coated with a polyanion (e.g. polystyrene sulfonate(PSS)) rendering the flexible substrate surface negatively charged. Thissurface is then coated with nanoparticles by incubating it with asolution containing positively charged nanoparticles, which may resultfrom a synthesis mechanism that require a positively charged detergent.

In some aspects, the plasmonic nanoparticles are attached to thesubstrate covalently via a bifunctional moiety containing a thiolreacting group. As an example, the first material is activated using anoxygen plasma treatment and reacted with a mercaptosilane (e.g.,(3-mercaptopropyl)trimethoxysilane or(3-mercaptopropyl)triethoxysilane). This creates a thiol-functionalizedflexible substrate that reacts with noble-metal nanoparticles to createa covalent linkage. In another nonlimiting example, the first materialis activated using oxygen plasma followed by a reaction with anaminopropylsilane (e.g., (3-aminopropyl)triethoxysilane or(3-aminopropyl)trimethoxysilane). This creates an amine-functionalizedflexible substrate that reacts with noble metal nanoparticles or isfurther modified with, for example, (3-mercaptopropionic acid NHSester). The NHS ester reacts to form a covalent bond with the amineleaving an unreacted thiol, which is then covalently bound to thenoble-metal nanoparticles.

Also disclosed herein are methods for enhancing the fluorescent signalfrom a fluorescent species. The method generally comprises: placing aplasmonic patch in proximity to the fluorescent species, exciting thefluorescent species using electromagnetic radiation of a predeterminedwavelength thereby generating the fluorescent signal, and detecting saidenhanced fluorescent signal. In some aspects, the plasmonic patch is asdescribed elsewhere herein.

As used herein, placing the patch in proximity to the fluorescentspecies is to be interpreted such that the patch is close enough to thefluorescent species in order to enhance the fluorescent signal emanatingtherefrom. In some aspects, this distance is greater than 0.5 nm,greater than 1 nm, greater than 2 nm, greater than 3 nm, greater than 5nm, greater than 7 nm, greater than 10 nm, greater than 12 nm, greaterthan 15 nm, or greater than 20 nm.

Initiation of a fluorescent signal is often initiated by exposing thefluorophore in the fluorescent species to electromagnetic radiation. Thesource of the electromagnetic radiation can be from any typical source,including, but not limited to, a tunable laser or an LED. The wavelengthof the radiation used to initiate the fluorescent signal is selectedbased on the excitation spectra of the fluorophore in the fluorescentspecies. The wavelength of the excitation source can be monochromatic orpolychromatic depending on the source of the electromagnetic radiation.For example, a tunable laser would be monochromatic while an LED sourcewould be polychromatic. The excitation spectra of the fluorophoredescribes the wavelength at which the fluorophore will absorb theradiation thereby initiating the fluorescent signal. The emissionspectra of the fluorophore describes the wavelength at which thefluorophore emits the fluorescent signal after excitation. Both theexcitation spectra and the emission spectra have a maximum value (i.e.,an inflection point). The difference between the excitation maximum andthe emission maximum in a fluorophore is called the Stokes shift. Insome instances, a fluorophore has two or more maxima where one is theglobal maxima and the other values are local maxima. In some aspects,the predetermined wavelength used to excite the fluorophore in thefluorescent species is the same as the excitation maximum for thefluorophore. The excitation maximum can be the global or local maximum.For the two wavelengths to be the “same”, this is within theexperimental error as determined by the capabilities of the radiationsource. The instrument that generates the energy of the predeterminedwavelength is tuned as closely as possible to the excitation maximum ofthe fluorophore in the fluorescent species.

In some aspects, the predetermined wavelength is selected so it is notthe same as the excitation maxima of the fluorophore in the fluorescentspecies. In some aspects, the predetermined wavelength is from about 300nm to about 2000 nm, about 400 nm to about 1000 nm, or about 450 nm toabout 800 nm. In some aspects, the predetermined wavelength is about 480nm, about 488 nm, about 532 nm, about 658 nm, or about 784 nm. In stillyet another aspect, the predetermined wavelength is about 300 nm, about350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about850 nm, about 900 nm, about 950 nm, or about 1000 nm. In still yetanother aspect, the predetermined wavelength is within ±5 nm of theexcitation maximum of the fluorophore in the fluorescent species. Instill yet another aspect, the predetermined wavelength is within ±10 nmof the excitation maximum of the fluorophore in the fluorescent species.In still yet another aspect, the predetermined wavelength is within ±15nm of the excitation maximum of the fluorophore in the fluorescentspecies. In still yet another aspect, the predetermined wavelength iswithin ±20 nm of the excitation maximum of the fluorophore in thefluorescent species. In still yet another aspect, the predeterminedwavelength is within ±25 nm of the excitation maximum of the fluorophorein the fluorescent species. In still yet another aspect, thepredetermined wavelength is within ±30 nm of the excitation maximum ofthe fluorophore in the fluorescent species. In still yet another aspect,the predetermined wavelength is within ±35 nm of the excitation maximumof the fluorophore in the fluorescent species. In still yet anotheraspect, the predetermined wavelength is within ±40 nm of the excitationmaximum of the fluorophore in the fluorescent species. In still yetanother aspect, the predetermined wavelength is within ±45 nm of theexcitation maximum of the fluorophore in the fluorescent species. Instill yet another aspect, the predetermined wavelength is within ±50 nmof the excitation maximum of the fluorophore in the fluorescent species.

In some aspects, the fluorescent species is a fluorescent dye. The dyeis any dye that generates a detectable fluorescent signal. Examplesinclude, but are not limited to, cyanine dyes (e.g., CY3, CY5), infrareddyes (e.g., 800CW), and alexa fluor dyes (e.g., alexa 488).

Exemplary embodiments of the plasmonic patch and methods for its use aredescribed above in detail. The plasmonic patch and methods describedherein are not limited to the specific embodiments described, butrather, components of apparatus, systems, and/or steps of the methodsare utilized independently and separately from other components and/orsteps described herein. For example, in some embodiments, the methodsare also used in combination with other polymers, nanostructures andbioassays, and are not limited to practice with only the apparatuses,systems, and methods described herein. Rather, the exemplary embodimentsare implemented and utilized in connection with many other systems.

Although specific features and applications of various embodiments ofthe disclosure may be shown in some drawings and not in others, this isfor convenience only. In accordance with the principles of thedisclosure, any feature illustrated herein may be referenced and/orclaimed in combination with any feature.

As various changes could be made in the above embodiments withoutdeparting from the scope of the disclosure, it is intended that allmatter contained in the above description and shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

Examples

Preparation of the Plasmonic Patch

Synthesis of Au@Ag-490. To synthesize Ag nanocubes, Au nanospheres witha diameter of 30 nm were employed as the seed. The 30 nm Au nanosphereswere in turn synthesized by a seed-mediated method. First, Au seeds weresynthesized by adding 0.6 mL of ice-cold NaBH₄ solution (10 mM) into asolution containing 0.25 mL HAuCl₄ (10 mM) and 9.75 mL CTAB (0.1 M)under vigorous stirring at room temperature for 10 min. The solutioncolor changed from yellow to brown indicating the formation of Au seed.Next, 0.25 mL of the seed solution was added to a growth solutioncontaining 10 mL of CTAC (cetyltrimethylammonium chloride, 0.2 M) and7.5 mL of ascorbic acid (0.1 M) under stirring. HAuCl₄ (10 mL, 0.5 mM)was added to the growth solution as a single addition, resulting in theformation of Au nanospheres with a diameter of 10 nm. The 10 nm Aunanospheres were centrifuged at 13,000 rpm for 30 minutes. For furthergrowth of Au nanoparticles to a diameter of 30 nm, a growth solutioncomprised of 30 mL CTAC (0.1 M) and 1.95 mL ascorbic acid (10 mM) wasprepared. To the resulting solution, 1.2 mL of 10 nm Au nanospheres(extinction 1.0) was added under stirring. A total 30 mL solution ofHAuCl₄ (0.5 mM) was added into the above mixture at the rate of 0.5ml/min under stirring. After the reaction was completed, the solutioncontaining 30 nm Au nanospheres was centrifuged at 8000 rpm for 10 minand redispersed into nanopure water to achieve a final extinction ˜1.2.

As-synthesized 30 nm Au nanospheres (1.5 mL) and 13.5 mL of CTAC (20 mM)were mixed under stirring at 60° C. for 20 min. Subsequently, 1.5 mL ofAgNO₃ (2 mM), 3.75 mL of CTAC (20 mM), and 0.7 mL of ascorbic acid (100mM) were added under stirring at 60° C., and the solution was left understirring for 4 h. After 4 h, the Au@Ag-490 nanocubes were centrifuged(8000 rpm) and redispersed into 7.5 mL aqueous solution of CTAC (20 mM)and stored in the dark until use. The average edge length of theAu@Ag-490 nanocubes was measured to be 48±1.4 nm from TEM images.

Synthesis of AuNR. AuNR was prepared by a seed-mediated method. Au seedwas synthesized by the method described above. For the synthesis ofAuNR-760, the growth solution was prepared by the sequential addition ofaqueous HAuCl₄ (0.01 M, 2 mL), CTAB (cetyltrimethylammonium bromide, 0.1M, 38 mL), AgNO₃ (0.01 M, 0.4 mL), and ascorbic acid (0.1 M, 0.22 mL)followed by gentle inversion to homogenize the solution. Subsequently,48 μL of the seed solution was added into the growth solution and leftundisturbed in dark for 12 hours. For AuNR-670, the growth solutioncontained HAuCl₄ (0.01 M, 2 mL), CTAB (0.1M, 40 mL), AgNO₃ (0.01 M, 0.4mL), HCl (1.0 M, 0.8 mL), and ascorbic acid (0.1 M, 0.32 mL). After theseed solution was diluted 50 times with nanopure water, 10 μL of thediluted seed solution was injected into the above growth solution andleft undisturbed in the dark for 12 hours. The obtained AuNR solutionswere then subjected to anisotropic oxidation by adding H₂O₂ (30 wt %).The oxidation process was monitored by measuring the extinction spectraof the AuNR solution. When the longitudinal LSPR wavelength reached thedesired value, the AuNRs were washed by two cycles of centrifugationwith 0.1M CTAB and finally redispersed in nanopore water. The finalextinction of AuNR-670 and AuNR-760 was adjusted to be ˜1.5.

Fabrication of plasmonic patch. Sylgard 184 (Dow Corning)polydimethylsiloxane (PDMS) elastomer was mixed at a 10:1 (base tocuring agent) ratio. The prepolymer (0.1 g) was spin coated at 3000 rpmfor 30 seconds on a polystyrene petri dish with a diameter of 3.5 cm.PDMS was then cured at 70° C. for 15 hours. Once cured, PDMS was treatedwith oxygen plasma for 3 mins and was immersed into 0.2% aqueouspoly(styrene sulfonate) (PSS) solution for 20 mins. PSS treatmentrendered a negative charge on the surface of PDMS film, whichfacilitates the absorption of positively charged plasmonic nanoparticlesthrough electrostatic interaction. For adsorbing the plasmonicnanostructures onto the modified PDMS surface, nanoparticle solution (1mL) was centrifuged and redispersed into a specific volume of nanopurewater (1.5 mL nanopure water for AuNR and 2 mL for Au@Ag-490). The PSStreated PDMS was incubated with the plasmonic nanoparticles for 15 hoursin the dark. Subsequently, PDMS was rinsed with nanopure water and driedwith nitrogen, leaving a surface with uniformly adsorbed plasmonicnanoparticles.

FIG. 34 illustrates one embodiment of the plasmonic patch disclosedherein. This embodiment comprises a rigid backing layer or backer 3410and reflective layer 3420 in addition to the flexible substrate 3430,spacer layer 3440 and plasmonic nanoparticles 3450. It also illustratesthe thickness of the spacer 3460.

Covalent linking of nanorods to PDMS. The cured PDMS substrate istreated with oxygen plasma for at least 3 minutes. The surface iscovered with an 8% (v/v) solution of (3-mercaptopropyl)trimethoxysilanedissolved in ethanol and incubated for one hour at room temperature. Thesolution is removed and the surface is washed with ethanol three times.For adsorbing the plasmonic nanostructures onto the modified PDMSsurface, the nanoparticle solution (1 mL) was centrifuged andredispersed into nanopure water (1.5 mL nanopure water for AuNR and 2 mLfor Au@Ag-490). The modified PDMS was incubated with the plasmonicnanoparticles for 15 hours in the dark. Subsequently, the PDMS substratewas rinsed with nanopure water and dried with nitrogen, thereby creatinga surface with uniformly adsorbed plasmonic nanoparticles.

Polymer spacer on plasmonic patch. APTMS (8 μL) and TMPS (0 to 8 μL)were added into 3 mL of phosphate buffered saline (1×PBS). The plasmonicpatch was incubated in the above solution for 2 hours. After 2 hours,the plasmonic patch was rinsed with PBS and nanopure water followed bydrying with nitrogen gas. For the plasmonic patch of AuNR-760, 0.25 μLTMPS and 8 μL of APTMS were added to 3 mL of PBS for 2 hours incubation.The film of AuNR-670 was modified with APTMS (8 μl APTMS in 3 mL PBS for2 hours incubation). These spacer conditions were used in the followingexperiments.

Fabrication of plasmonic patches having different densities of AuNR-758nanorods. The influence of the density of the plasmonic nanostructureson the fluorescence enhancement efficiency was tested. AuNR-758 wereutilized to probe the effect of density (FIG. 30A). To obtain plasmonicpatches with varying density of nanostructures, the PDMS films wasexposed to AuNR solutions of varying concentrations. The extinctionspectra and SEM images obtained from these plasmonic patches illustratethe variation in the density of the nanostructures, directlycorresponding to the concentration of AuNR solution (FIG. 30B, 30C). SEMimages of the plasmonic patches revealed AuNR densities on theseplasmonic patches to be 13±2/μm², 16±2/μm², 45±3/μm², 62±4/μm², 72±4/μm²and 93±4/μm² (FIG. 30C). The color of plasmonic patches and extinctionintensity of the LSPR band intensified gradually in a density-dependentmanner (FIG. 30B). To quantify the fluorescence enhancement efficiency,the plasmonic patches with varying AuNR density were transferred ontothe bottom of polystyrene wells (in a 96-well plate) coated with 800CW(tagged to streptavidin). The plasmonic patches exhibited a densitydependent-fluorescence enhancement, with higher density of plasmonicnanostructures resulting in higher enhancement (FIG. 30D, 30E). Theenhancement rapidly increased up to a density of ˜45/μm² and remainedvirtually unchanged for higher densities.

Fabrication of plasmonic patch having different size nanorods. Using theprocedures described above, different size nanorods were prepared (FIGS.31A and 32A) and incorporated into the plasmonic patch at a constantdensity of nanorods. Extinction coefficients were normalized (FIG. 32B)such that the AuNRs exhibited a similar LSPR after incorporation intothe PDMS film (FIG. 31D). As illustrated in FIGS. 31B and 31C, the sizeof the AuNRs affects the enhancement of the fluorescent signal with amaximum enhancement for AuNRs measuring about 113 nm by 38 nm.

Fluorescence Enhancement Using the Plasmonic Patch.

Aldehyde activation of silicon and glass substrates. Glass and siliconsubstrates were cleaned using Piranha solution (3:1 concentratedsulfuric acid to 30% hydrogen peroxide solution) followed by thoroughrinsing with nanopure water and drying with nitrogen gas. A mixture of0.25 mL of (3-aminopropyl)triethoxysilane (APTES) and 0.25 mL ofnanopure water was added into 4.5 mL pure ethanol followed by mixing.Cleaned silicon and glass substrates were incubated in the APTESsolution for 2 hours. The substrates were subsequently rinsed withnanopure water and ethanol followed by drying under nitrogen gas.APTES-modified substrates were incubated in 2.5% glutaraldehyde (GA)solution (PBS, pH=7.4) for 2 hours. The substrates were rinsed with PBSfollowed by nanopure water to remove excess GA. The aldehyde activatedsubstrates react with primary amines of the protein to form Schiff'sbase linkage.

Plasmonic patch enhanced fluorescence. 96-well plate (polystyrene),nitrocellulose membrane (average pore size of 0.22 μm), aldehydeactivated glass and silicon substrates were incubated in 800CW labeledanti-mouse IgG (10 ng/mL in PBS) in the dark for 8 hours. The substrateswere then rinsed with PBS and nanopure water. Plasmonic patch withAuNR-760 was cut into a round shape with a diameter of 4 mm. For thesilicon substrate, the film was directly applied on top of the wetsurface, and the sample was scanned after 20 mins. For the othersubstrates, a reflective flexible film (PET film Novele IF-220(Novacentrix)) coated with 50 nm of gold layer using thermo-evaporator)was added on the top of plasmonic patch. Bottom reading, orepifluorescence, mode was adopted for these substrates due to theiroptical transparency, and the reflective film increased the signal byabout 2-fold. All the fluorescent signals were obtained by LICOR OdysseyCLx imager.

ELISA. Duoset ELISA from R&D systems (DY1750B, DY1757) were employed forthe quantification of Neutrophil Gelatinase Associated Lipocalin (NGAL)and Kidney Injury Molecule-1 (KIM1), respectively. 96-well plasticplates were first coated with capture antibodies (For NGAL: 2 μg/mL inPBS; for KIM1: 4 μg/mL in PBS) through overnight incubation at roomtemperature, followed by blocking with 300 μL reagent diluent (PBScontaining 1% BSA, 0.2 μm filtered). After three times washing with PSBT(0.05% Tween 20 in PBS), 100 μL of serial diluted standard samples aswell as patients' urine samples (10-fold dilution for KIM1, 40-folddilution for NGAL) were added into different wells (all dilutions usingreagent diluent). The plate was covered with an adhesive strip andincubated at room temperature for 2 hours. After washing three timeswith PBS, the plate was incubated with biotinylated detection antibodies(For NGAL: 25 ng/mL in reagent diluent; for KIM1: 50 ng/mL in reagentdiluent) at room temperature for 2 hours, washed another three timeswith PBS, incubated with HRP labeled streptavidin (200-fold dilutionusing reagent diluent) for 20 mins, and washed three times with PBS. 100μL of substrate solution (1:1 mixture of Color Reagent A (H₂O₂) andColor Reagent B (Tetramethylbenzidine) (R&D Systems, Catalog #DY999))was added to each well and the reaction was stopped by adding 50 μL ofH₂SO₄ (2 N) (R&D Systems, Catalog #DY994) after 20 mins. Optical densityof each well was determined using a microplate reader set to 450 nm.

Fluorescence-linked immunosorbent assay with plasmonic patch.Fluorescence-linked immunosorbent assay was first implemented using96-well plates with glass bottom (Cellvis). The glass surface of eachwell was treated to achieve aldehyde functionality using the methoddescribed above. Subsequent procedures were identical with ELISA untilthe streptavidin binding step. Instead of HRP-labeled streptavidin, 100μL of dye-labeled streptavidin (800CW or LT680 (LICOR)) was diluted to afinal concentration of 50 ng/mL using reagent diluent and added to eachwell followed by a 20-min incubation. Plasmonic patch was subsequentlytransferred to each well of the 96-well plate using the methodintroduced above. The LICOR Odyssey CLx scanner was used to scan the96-well plate using 800 nm channel (intensity of 3.0, resolution of 84μm, scanning height of 2 mm) and 700 nm channel (intensity of 3.5,resolution of 84 μm, scanning height of 2 mm). For thefluorescence-linked immunosorbent assay performed using plastic bottom96-well plates, the procedure remained the same except the omission ofsurface modification steps. While scanning, the height was set to 4 mmfor the plastic 96-well plate. All the fluorescent signals were analyzedby calculating the average intensity of the center (2 mm diameter)within each well.

Fluorescence enhancement on protein microarray. Commercialized proteinmicroarray chip kits were purchased from RayBiotech (Custom G-SeriesAntibody Array, AAX-CUST-G). Antibodies were printed on a glass slidewith four subarrays available per slide. The slide was blocked byblocking buffer for 30 mins. Urine samples were diluted twice usingblocking buffer and 90 μL of the diluted samples was added to eachsub-well of the microarray chip followed by a two-hour incubation atroom temperature. The chip was then washed thoroughly with wash buffer.Seventy microliters of biotin-conjugated anti-cytokines was added toeach subarray and the chip was incubated at room temperature. After twohours, the chip was washed, and 70 μL of streptavidin-800CW (100 ng/mLin blocking buffer, LICOR) was added and the plate was incubated in thedark for 20 mins. The chip was washed thoroughly with wash buffer thennanopure water and dried under nitrogen gas. The glass chip was scannedby LICOR Odyssey CLx scanner using 800 nm channel (intensity of 2,resolution of 21 μm, scanning height of 1 mm). Plasmonic patch ofAuNR-760 was cut into 1×1 cm² and applied on the top of each subarrayfollowed by attachment of gold coated reflective film of the same size.The chip was rescanned using the same settings. Median background signalwas adopted for analyzing the spot intensity.

Plasmonic patch material characterization. A thin polydimethylsiloxane(PDMS) layer (˜30 μm thick) is employed as the “film” material owing toits high mechanical flexibility (elastic modulus ˜1 Mpa) (FIG. 1),optical transparency (>95% transmittance within the wavelength range of400 to 900 nm), excellent processability, and low cost. The elastomericnature of PDMS enables conformal contact (down to atomic level) of thefilm with diverse surfaces, which helps to improve the fluorescenceenhancement as the enhanced electromagnetic field of the plasmonicnanostructures is limited to a short distance. The plasmonic patch istailored for a specific fluorophore by maximizing the overlap betweenthe localized surface plasmon resonance (LSPR) of the nanostructures andthe optical absorption (excitation source) of the fluorophore to achievethe highest enhancement. As representative examples, three differentfilms were fabricated using three different nanostructures: (i) goldcore-silver shell nanocubes (Au@Ag nanocubes) with LSPR wavelength at490 nm (Au@Ag-490 henceforth); gold nanorods (AuNR) with longitudinalLSPR wavelength at (ii) 670 nm (AuNR-670 henceforth) and (iii) 760 nm(AuNR-760 henceforth) (FIG. 2). SEM images indicate a highly uniformdistribution of the plasmonic nanostructures on the PDMS film with nosign of aggregation or patchiness (FIG. 3), which ensures a nanoscaleconformal contact between the plasmonic patch and surface of interest.Extinction spectra obtained from the plasmonic patches further validatethe absence of aggregates (FIG. 4). The flexible plasmonic patchesexhibit distinct and uniform color corresponding to the LSPR wavelengthof the nanostructures. The three films described above were designed forfluorescein isothiocyanate (FITC) (Au@Ag-490), LT680 (AuNR-670), and800CW (AuNR-760) as a representative fluorophore. Transfer of thecorresponding plasmonic patches to silicon surfaces coated with FITC,LT680, and 800CW resulted in a uniform enhancement of fluorescence fromthese surfaces (FIG. 5). Additionally, the transfer process is easy, anddoes not require special training for users to implement (FIG. 6). Thefluorescence intensity in the presence of plasmonic patch was found to˜50-fold higher compared to that obtained from an unenhanced surfaceunder identical illumination conditions (FIG. 7). Apart from silicon,the plasmonic patch was also applied to glass, nitrocellulose, andpolystyrene (common material for microtiter plates) surfaces, which areextensively employed in various fluorescence-based detection,quantitative sensing and imaging techniques. The excellent conformity ofthe plasmonic patch with all of the above materials resulted in largefluorescence enhancement of the dye deposited on these surfaces. Theintensity cross-section profiles from these different materialsdemonstrated ˜80-fold enhancement in the fluorescence from the regionswith plasmonic patch (center) compared to unenhanced regions (periphery)(FIG. 8).

Distance-dependent fluorescence enhancement and spacer layer. It isknown that the evanescent nature of the enhanced electromagnetic fieldat the surface of the plasmonic nanostructures results in a highlydistance-dependent enhancement of Raman scattering and fluorescence atthe surface of the plasmonic nanostructures. When fluorophores arebrought in direct contact (or in extreme proximity) with plasmonicnanostructures, non-radiative energy transfer between the fluorophoreand metal surface results in fluorescence quenching. On the other hand,increasing the distance between the fluorophores and metalnanostructures results in a decrease in the enhancement due to the decayin the electromagnetic field from the surface of the nanostructures.Taken together, an optimal distance between the metal surface andfluorophore is tuned to optimize the fluorescent enhancement for eachindividual assay.

In one example, optimization of the distance between plasmonicnanostructures and fluorophores was done. A polysiloxane copolymer filmwas formed on the surface of the plasmonic patch as a spacer layer (FIG.9). Trimethoxypropylsilane (TMPS) and (3-aminopropyl) trimethoxysilane(APTMS) were copolymerized onto the plasmonic patch comprised ofAuNR-760. The two monomers underwent hydrolysis and condensationyielding an amorphous copolymer layer (FIG. 10). Increasing thethickness of the spacer layer results in a gradual red shift of thelongitudinal LSPR wavelength of AuNR owing to the increase in therefractive index of the medium surrounding the nanostructures (FIG. 11).With increase in the spacer layer thickness, a steep increase in thefluorescence enhancement efficacy of 800CW followed by relativelyshallow reduction was observed (FIGS. 12 and 13). Atomic forcemicroscopy (AFM) images depicted the morphology change of the plasmonicpatch after the formation of polysiloxane layer, which further confirmedthe uniform deposition of polymer spacer onto the AuNR (FIG. 14).Plasmonic patches with an optimal spacer layer thickness were testedherein.

Plasmonic patch enhanced fluoroimmunoassays. A typical sandwichfluoroimmuno-assay involves the following major steps (i) capture of thetarget antigen by an immobilized antibody; (ii) binding of thebiotinylated detection antibody to the captured antigen; and (iii)binding of a fluorescently-labeled streptavidin (FIG. 15). To test theuniversality and ease of use of the plasmonic patch disclosed herein, afluoroimmunoassay in a heterogeneous, solid phase format by using a96-well microtiter plate was implemented. This is a standard assayformat extensively employed in bioanalytical research and clinicaldiagnostics (FIG. 20).

KIM-1 and NGAL Fluoroimmunoassay. Two early stage biomarkers for acutekidney injury (AKI) and chronic kidney disease (CKD) were examined,kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associatedlipocalin (NGAL), as representative examples for probing the efficacy ofthe plasmonic patch in improving bioanalytical parameters offluoroimmunoassays. The assays were implemented on a 96-well plate witha glass bottom. In the MM-1 immunoassay, LT680 was the fluorescence tag,and the plasmonic patch comprised AuNR-670. To probe the enhancement insensitivity and LOD, serial dilutions of KIM-1 of known concentrations(5 ng/mL to 500 fg/ml) in phosphate buffered saline (PBS) with 1% bovineserum albumin (BSA) were employed as standards. Fluorescence imagesobtained after applying the plasmonic patch revealed a large enhancementin fluorescent intensity compared to that obtained before applying theplasmonic patch (FIG. 16). The fluorescence signal from the unenhanced(pristine) spots was detectable only for the two highest concentrations(5 and 0.5 ng/ml) (FIG. 16, left and middle images). On the other hand,fluorescence signal with plasmonic patch could be detected down to 500fg/mL (FIG. 16). The concentration-response plot revealed a 36-foldenhancement in the fluorescence intensity with plasmonic patch comparedto the unenhanced signal (FIG. 16). The LOD (3σ) of the unenhanced andplasmon-enhanced MM-1 assays were determined to be 140 μg/mL and 0.5μg/mL, respectively, which represents a 280-fold improvement in the LOD.Consequently, the enhanced MM-1 assay exhibited three orders ofmagnitude higher dynamic range compared to the unenhanced assay. Thefluorescence signal after application of plasmonic patch exhibitedexcellent stability even after eight weeks storage in the dark (FIG.17). To demonstrate the broad applicability of plasmon-enhancedfluoroimmunoassay, NGAL was tested as another representative example.800CW (conjugated to streptavidin) was the fluorescence label todemonstrate the tunability of the plasmonic patch. Following thetransfer of the plasmonic patch, a fluorescence enhancement up to 103times and a ˜100-fold lower LOD compared to the unenhanced NGAL assaywas observed (FIG. 18). Consistently, the NGAL assay implemented oncommon 96-well plate with a plastic bottom (instead of glass bottom)also exhibited large fluorescence enhancement in the presence ofplasmonic patch (FIG. 19), which further validates the plasmonic patchas a substrate material-agnostic technology.

Enzyme-linked immunosorbent assay (ELISA) is widely employed in clinicaland research settings and often considered a “gold standard” for proteinbiomarker detection and quantification. The performance of theplasmon-enhanced fluoroimmunoassay with ELISA using MM-1 as arepresentative biomarker was tested. The LLOD of plasmon-enhancedfluoroimmunoassay was found to be ˜30-fold lower (0.5 pg/ml) compared tothat of ELISA (15.6 pg/ml) (FIGS. 16 and 20). Notably, the dynamic rangeof the enhanced fluoroimmunoassay spanned five log orders of KIM-1concentration, while the dynamic range of ELISA was only 2.5 log ordersof KIM-1 concentration (FIGS. 16 and 20). The higher dynamic range ofthe enhanced fluoroimmunoassay is expected to enable the quantificationof a wider range of biomarker concentrations in human urine samples asdescribed below.

Following the successful demonstration of the plasmonic patch enhancedfluoroimmunoassay, urine samples from patients and self-describedhealthy volunteers were analyzed to determine the concentrations ofKIM-1 and NGAL. In order to demonstrate the wide applicability of thetechnique, both glass (KIM-1) and plastic (NGAL) bottom 96-well plateswere used. The urine samples were diluted with 1% BSA in PBS to minimizeconfounding from inter-individual differences in urine pH and solutecontent. For KIM-1 (10-fold dilution) and NGAL (40-fold dilution), theplasmon-enhanced fluoroimmunoassay exhibited a dramatic increase in thefluorescence compared to the unenhanced fluoroimmunoassay (FIG. 21(KIM-1) and (NGAL)). The enhanced fluorescence signal was employed toquantify the biomarker concentration in the urine samples. StandardELISA was also used to determine KIM-1 and NGAL concentrations in thehuman urine samples. The concentration of the biomarker in urinedetermined by the above three assays (unenhanced and enhancedfluoroimmunoassays, and ELISA) is compared in FIG. 22. The unenhancedfluoroimmunoassay was not able to detect KIM-1 (FIG. 22A) or NGAL (FIG.22B) in any of the human urine samples. In stark contrast,plasmon-enhanced fluoroimmunoassay was able to quantify both KIM-1 andNGAL concentrations in all human urine samples, some of which were evenlower than the LOD of ELISA. For the samples that were quantifiableusing both ELISA and enhanced fluoroimmunoassay, the concentration ofthe biomarker determined using the enhanced fluoroimmunoassay showedexcellent agreement with that determined using “gold standard” ELISA forboth KIM-1 (r²=0.934) and NGAL (r²=0.998) (FIGS. 23A and 23B).

Biomarker concentrations in the human urine samples were determined byaccounting for the dilutions in each of the assays, and the results aretabulated in Table 1. The standard metric of kidney function is theestimated Glomerular Filtration Rate (eGFR), determined from serumcreatinine concentration. eGFR decreases below 90 (mL/min) as the kidneyfunction declines. The two urine biomarkers can provide diagnostickidney disease information beyond that of eGFR. NGAL and KIM-1concentrations in healthy humans are <20 ng/mL and <1 ng/mL,respectively. In acute kidney injury, NGAL exceeds 100 ng/mL. Takingpatient #24 and #37 as examples, while their eGFR levels (153 mL/min and90 mL/min) are within the normal range, NGAL and KIM-1 concentrationswere significantly higher, indicating a high risk of chronic kidneydisease (#24) and acute kidney injury (#37). Notably, for diabetics,their eGFR levels tend to increase to 150 mL/min followed by asignificant decrease (down to 30 mL/min) with time. The higher eGFR ofpatient #24 and #37 and slightly elevated KIM-1 and NGAL concentrationsmay be due to the patient being diabetic, which is a risk factor forchronic kidney disease (Table 1).

TABLE 1 Patient # Age Sex Diabetes eGFR KIM-1 NGAL 1 26 F − — 118 0.5624 31 F + 153 280 29.30 25 61 M + 87 1520 38.00 26 80 M − — 136 6.80 2765 M − 131 80 2.10 28 52 F + — 2800 6.40 29 66 F + 66 1030 18.30 30 59 M− 76 480 13.70 37 68 F + 90 2220 110.00 403 63 M − — 110 0.78 404 56 F −— 220 18.81

Application of plasmonic patch on a protein microarray. To demonstrateapplicability of the plasmonic patch in enhancing the sensitivity ofimmuno-microarrays, a microarray of antibodies of biomarkers for kidneyinjury were tested to assess the performance of plasmonic patch in amultiplexed, high throughput biosensing platform (FIG. 24). Thismicroarray comprised eight capture antibodies corresponding to AKI andCKD protein biomarkers, printed in duplicates on a glass slide isolatedby a plastic frame, with a feature diameter of 150 μm. Biotinylated IgGsof three gradient concentrations were printed in duplicates as positivecontrols (FIG. 25, left schematic showing the protein layout on themicroarray). Six human urine samples were diluted 2-fold using blockingbuffer and added to each sub-well on the slide. The captured biomarkerproteins were exposed to a biotinylated detection antibody cocktailfollowed by exposure to 800CW-labeled streptavidin. A conventionalmicroarray procedure ends here by analyzing the localized fluorescentsignal on the respective antibody spot. The enhanced assay demonstratedhere involves the addition of a 1×1 cm² plasmonic patch modified withAuNR-760 on top of each array.

The fluorescence map from a single sample (patient #81, FIG. 25, rightpanels) is informative. Apart from large enhancement of the weakfluorescence of albumin, cystatin C, β2 microglobulin (Beta 2M), andNGAL in the unenhanced microarray, the plasmonic patch enabled thedetection and quantification of analytes that were not detected by theconventional method (gray boxes in FIG. 25). These new analytes aretissue inhibitor of metalloproteinases 2 (TIMP-2), KIM-1, andinsulin-like growth factor-binding protein 7 (IGFBP-7), which arespecific and important biomarkers for early detection of acute kidneyinjury. In all samples tested, the plasmonic patch enhanced thefluorescence signals of the microarray exposed to urine samples (FIG.26). Quantitative measurement of antibody spot intensity from the urineof six individuals showed 20 to 137-fold increase in the fluorescence ofseveral analytes, and the ability to detect other analytes only with theenhancement from plasmonic patch (FIG. 27, [+] mark indicating biomarkeronly detected with plasmonic patch). Comparison between the unenhancedand plasmonic patch enhanced fluorescence heat maps from the six donorsfurther revealed the high signal-to-noise ratio and a broadened dynamicrange (FIG. 28).

cTnl Fluoroimmunoassay. The fluoroimmunoassay was implemented using96-well plates having a plastic bottom. Briefly, 100 μL of standardsolutions with different cTnI concentrations or patient samples werefirst added into appropriate wells. The plate was sealed and gentlyshaken for 2.5 hours at room temperature. The solution was discarded,and the wells were washed 4 times with washing solution. Subsequently,100 μL of biotinylated antibody solution (according to vendor'sprotocol) was added into each well and incubated for 1 hour at roomtemperature. Next, 100 μL of dye-labeled (800CW) streptavidin (50 ng/mL)was added to each well followed by 30 min incubation. Finally, theplasmonic patch was transferred onto each well of the 96-well plate,followed by the addition of a reflective layer on the top. A LICOROdyssey CLx scanner was used to scan the 96-well plate.

As shown in FIGS. 29A to 29D, the use of the plasmonic patch greatlyenhanced the fluorescent signal exhibited by 800CW. In the top half ofFIG. 29A, no detectable signal is observed without the plasmonic patchwhile a greatly enhanced signal is seen in the lower half of FIG. 29A.FIG. 29B illustrates the effect of nanostructure density on the signalenhancement. FIGS. 29C and 29D illustrate that the sensitivity isgreatly enhanced to at least as low as 1 pg/mL.

IL-6 Fluoroimmunoassay. Using a modification of the fluoroimmunoassaysdescribed above, an IL-6 fluoroimmunoassay was done to determine theLLOD and amount of enhancement of the fluorescent signal generatedtherein. As shown in FIGS. 33A and 33B, the lower limit of detection wasdetermined to be about 333-fold lower than the same assay without theuse of the plasmonic patch. Concentrations as low as 0.006 pg/mL weredetected and quantified where the LLOD of this assay when conductedwithout a plasmonic patch is approximately 2.0 pg/mL.

This written description uses examples to describe the disclosure,including the best mode, and also to enable any person skilled in theart to practice the disclosure, including making and using any devicesor systems and performing any incorporated methods. The patentable scopeof the disclosure is defined by the claims, and may include otherexamples that occur to those skilled in the art. Such other examples areintended to be within the scope of the claims if they have structuralelements that do not differ from the literal language of the claims, orif they include equivalent structural elements with insubstantialdifferences from the literal languages of the claims.

What is claimed is:
 1. A plasmonic patch for enhancing a fluorescentsignal from a fluorescent species, the patch comprising: a flexiblesubstrate comprising a first material, a plasmonic nanostructure, and aspacer comprising a second material; wherein the plasmonic nanostructureis disposed on a first surface of the flexible substrate, the spacer isdisposed on the first surface of the flexible substrate and covers theplasmonic nanostructure; the fluorescent species has an excitationwavelength (λ_(ex)) and an emission wavelength (λ_(em)), the plasmonicnanostructure has a localized surface plasmon resonance (LSPR)wavelength (λ_(LSPR)), and a difference between the LSPR wavelength andthe excitation wavelength is |Δλ|.
 2. (canceled)
 3. The plasmonic patchaccording to claim 1, wherein the first material and the second materialindependent of each other are functionalized with siloxane or selectedfrom the group consisting of an elastomeric polymer,polydimethylsiloxane (PDMS), ECOFLEX®, SILBIONE®, polyethyleneterephthalate (PET), polyurethane (PU), polyethylene naphthalate (PEN),polyimide (P1), polybutadiene, polyisoprene,(3-aminopropyl)trialkoxysilane, (3-aminopropyl)triaryloxysilane,(3-aminopropyl)triethoxysilane (APTES), (3-aminopropyl)trimethoxysilane(APTMS), trimethoxy(propyl)silane (TMPS),(3-mercaptopropyl)trimethoxysilane (MPTMS), polyamine,polymethylmethacrylate (PMMA), polydopamine, polyvinylpyrrolidone (PVP),polyvinyl alcohol (PVA), polyolefin, polyimide, polyimide, proteins,silk, cellulose, polyelectrolytes, peptoids and combinations thereof.4-7. (canceled)
 8. The plasmonic patch according to claim 1, wherein thespacer has a thickness of from 0.5 to 20 nm. 9-10. (canceled)
 11. Theplasmonic patch according to claim 1, the patch further comprising: abacking layer, wherein the backing layer has a Shore 00 hardness that isgreater than a Shore 00 hardness of the first material, and wherein theflexible substrate is disposed between the spacer and the backing layer;and a reflective layer, wherein said reflective layer is disposedbetween the backing layer and the spacer.
 12. (canceled)
 13. Theplasmonic patch according to claim 11, wherein the backing layercomprises a material selected from the group consisting of glass,plastic, a polymer, aluminum, mylar, RE1SIAR™, and combinations thereof.14-15. (canceled)
 16. The plasmonic patch according to claim 1, whereinthe plasmonic nanostructurec is selected from the group consisting ofnanotubes, nanorods, nanocubes, nanospheres, nanostructures with sharptips, nanostars, hollow nanostructures, nanocages, nanorattles,nanobipyramids, nanoplates, self-assembled nanostructures, bowtieantennae, nano islands, and combinations thereof.
 17. The plasmonicpatch according to claim 16, wherein the plasmonic nanostructurecomprises nanorods having a length of from about 25 nm to about 2000 nmand a diameter of from about 4 nm to about 100 nm.
 18. (canceled) 19.The plasmonic patch according to claim 16, wherein the plasmonicnanostructurec comprises nanocubes, nanocuboids, or a combinationthereof, wherein the nanocubes and/or nanocuboids have an average edgelength of from about 25 nm to about 1000 nm.
 20. (canceled)
 21. Theplasmonic patch according to claim 1, wherein the plasmonicnanostructures comprises a plasmonic material selected from the groupconsisting of gold, silver, copper, aluminum, a semiconductor, andcombinations thereof. 22-25. (canceled)
 26. The plasmonic patchaccording to claim 1, wherein a density of the nanostructures on theflexible substrate is from 1/μm² to 200/μm².
 27. (canceled)
 28. Theplasmonic patch according to claim 1, wherein the |Δλ| is 50 nm or less.29. (canceled)
 30. A method for enhancing a fluorescent signal of afluorescent species, the method comprising: placing a plasmonic patch inproximity to the fluorescent species, exciting the fluorescent speciesusing electromagnetic radiation of a predetermined wavelength therebygenerating an enhanced fluorescent signal, and detecting said enhancedfluorescent signal; wherein the plasmonic patch comprises: a flexiblesubstrate comprising a first material, a plasmonic nanostructure, and aspacer comprising a second material; wherein the plasmonicnanostructures is disposed on a first surface of the flexible substrate,the spacer is disposed on the first surface of the flexible substrateand covers the plasmonic nanostructure; the fluorescent species has anexcitation wavelength (λ_(ex)) and an emission wavelength (λ_(em)), theplasmonic nanostructure has a localized surface plasmon resonance (LSPR)wavelength (λ_(LSPR)), and a difference between the LSPR wavelength andthe excitation wavelength is |Δλ|.
 31. (canceled)
 32. The methodaccording to claim 30, wherein the predetermined wavelength is within±20 nm of the λ_(ex). 33-35. (canceled)
 36. The method according toclaim 30, wherein the first material and the second material independentof each other are functionalized with siloxane or selected from thegroup consisting of polydimethylsiloxane (PDMS), ECOFLEX®, SILBIONE®,polyethylene terephthalate (PET), polyurethane (PU), polyethylenenaphthalate (PEN), polyimide (P1), polybutadiene, polyisoprene,(3-aminopropyl)trialkoxysilane, (3-aminopropyl)triaryloxysilane,(3-aminopropyl)triethoxysilane (APTES), (3-aminopropyl)trimethoxysilane(APTMS), trimethoxy(propyl)silane (TMPS),(3-mercaptopropyl)trimethoxysilane (MPTMS), polyamine,polymethylmethacrylate (PMMA), polydopamine, polyvinylpyrrolidone (PVP),polyvinyl alcohol (PVA), polyolefin, polyimide, polyimide, proteins,silk, cellulose, polyelectrolytes, peptoids and combinations thereof.37-40. (canceled)
 41. The method according to claim 30, wherein thespacer has a thickness of from 0.5 to 20 nm. 42-43. (canceled)
 44. Themethod according to claim 30, the patch further comprising: a backinglayer, wherein the backing layer has a Shore 00 hardness that is greaterthan a Shore 00 hardness of the first material, and wherein the flexiblesubstrate is disposed between the spacer and the backing layer; and areflective layer, wherein said reflective layer is disposed between thebacking layer and spacer. 45-48. (canceled)
 49. The method according toclaim 30, wherein the plasmonic nanostructures is selected from thegroup consisting of nanotubes, nanorods, nanocubes, nanospheres,nanostructures with sharp tips, nanostars, hollow nanostructures,nanocages, nanorattles, nanobipyramids, nanoplates, self-assemblednanostructures, bowtie antennae, nano islands, and combinations thereof.50-53. (canceled)
 54. The method according to claim 30, wherein theplasmonic nanostructures comprises a plasmonic material selected fromthe group consisting of gold, silver, copper, aluminum, a semiconductor,and combinations thereof. 55-57. (canceled)
 58. The method according toclaim 30, wherein a density of the nanostructures on the flexiblesubstrate is from 1/μm² to 200/μm².
 59. (canceled)
 60. The methodaccording to claim 30, wherein the |Δλ| is 50 nm or less.